Abstract Background: Recently, several publications demonstrated that the miR-34 family of microRNAs may have a role in carcinogenesis and that they can be regulated by the p53 tumor suppressor gene. Because of these findings, we examined the expression of miR-34a, −34b and −34c in colon adenocarcinomas to determine if their expression was associated with colon cancer or p53 status of tumors. Methods: RNA was isolated from flash frozen tissue from a cohort of 159 American and 113 Chinese colon cancer patients. qRT-PCR was used to measure the expression of miR-34a, −34b, −34c and U6B from tumors and paired noncancerous tissues for each patient. p53 status was evaluated by immunohistochemistry for patients in both cohorts and by DNA sequencing for the Chinese cohort. The expression levels of miR-34a, −34b and −34c were analyzed for associations with diagnosis, TNM staging, cancer-specific mortality and P53 status of the tumors. Affymetrix mRNA array data was available for 69 members of the American cohort and this data was used to correlate miR-34 expression with mRNA expression and perform Ingenuity Pathway Analysis to indentify pathways that are associated with miR-34 expression. Results: miR-34a, −34b and miR-34c expression levels were significantly elevated in tumors in each cohort. The expression of miR-34b and miR-34c were significantly higher in more advanced staged tumors and this was observed independently in each cohort. Elevated miR-34b and miR-34c levels were also associated with worse cancer specific mortality (hazard ratio, 1.6; 95% confidence interval, 1.1-2.5). We found no evidence that expression levels of miR-34a, miR-34b or miR-34c were associated with p53 status of the tumors. Ingenuity Pathway Analysis on genes correlated with miR-34 expression indicated that genes with known roles in colon carcinogenesis were enriched in this gene set. The top canonical pathways to be enriched were mitochondrial dysfunction and oxidative phosphorylation. Conclusions: Increased miR-34a, miR-34b and miR-34c were observed in colon tumors from two independent cohorts. Elevated miR-34b and −34c expression were observed in more advanced tumors and high levels of these microRNAs were associated with worse survival. These results suggest a role for miR-34 in the progression of colon cancer. Surprisingly, we found no evidence for an association of P53 with any of the miR-34 family members. This suggests that the in vitro mechanisms demonstrating a connection between p53 and miR-34 expression may not be relevant to colon cancer. To address what may be regulating miR-34 expression, we performed pathway analysis and found evidence that the mitochondrial dysfunction and oxidative phosphorylation pathways are associated with miR-34 expression indicating that oxidative stress, independent of P53 status, may be an important regulator of miR-34 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3053.